The Effects Of Metformin And RIP140 On The AMPK-Mediated Anti-Tumor Classical Pathway

Objective:Current studies have shown that metformin have the potential to induce tumor cell apoptosis and inhibit proliferation.As a transcriptional co-repre-ssor factor,RIP140 has been reported many times and tumor development as well as insulin resistance.To investigate the relationship between the RIP140 and the potential mechanism of metformin’s inhibition of proliferation,and induction of apoptosis effects on liver cancer cell.Methods:1、Firstly,calculate IC50 of Metformin.Then human HepG2 cells were cultured in vitro and treated with different concentration of metformin(0、5.0、10.0、20mmol/l)for nearly 48 hours.Changes in cell proliferatio and apoptosis rate were evaluated by CCK-8 assay and flow cytometry,the expression level of protein of p-AMPK、AMPK、PGC-1α、RIP140 in each experimental group is detected by Western blot.2、The HepG2 RIP140 gene over-expression and downregulation models were constructed by lipofection and validated by real time polymerasechain reaction(PCR)and Western blot.The proliferatio and apoptosis were evaluated by flow cytometry.3、Establish the downregulated RIP140 cell model with metformin-treated,the protein expression levels of p-AMPK 、total AMPK、RIP140、p53、Bax、Bcl-2、cleaved-caspase 3were detected by Western blot,and apoptosis rate were evaluated by flow cytometry.Results:1、Human HepG2 cells were cultured in vitro and treated with different concentration of metformin(0、5.0、10.0、20mmol/l)for nearly 48 hours.1)IC50 of Metformin,s effect on HepG2 cell is(22.08±4.50)mmol/l.2)the results of CCK-8 showed:compared with 0mmol/l group,the 5mmol/l group proliferation decreased,but there is no statistical significance(P>0.05);the proliferation decrease of others groups are significant statistically(P<0.01).3)flow cytometry results showed:the apoptosis was induced in the metformin-treated groups compared the control group in a dose dependent manner P<0.05.While there is no statistical significance between 5mmol/l group and 10 mmol/l group(P>0.05.4)Western blot results showed:with the increasing concentration of metformin,the protein expression level of p-AMPK/AMPK、PGC-1α、RIP140 is higher and higher;among them,the level of p-AMPK/AMPK in 5mmol/l group is higher with on statistical significance vs 0mmol/l group(P>0.05);the level of PGC-1α in 20mmol/l group is higher vs10mmol/l group on statistical significance(P>0.05);the level of RIP140 protein in any group is higher and higher denpending on the dose of the concenteation of metformmin(P<0.01).2、The HepG2 RIP140 gene over-expression and downregulation models were successfully constructed(P<0.01).Compared with the normal group and the empty plasmid group,the over-expression of RIP140 remarkably inhibited HepG2 cell proliferation and promote apoptosis(P<0.05).While,the downregulation of RIP140 shows the just opposite results(P<0.05).3、Establish the downregulated RIP140 cell model with metformin-treated,evaluated the apoptosis rate and the certain protein level:1)flow cytometry results showed:the apoptosis rate of Metformin group is higher than control group on statistical significance(P<0.01);RIP140-shRNA+Metformin group showed the same vs control group(P<0.05),but decreased vs the Metformin group without statistical significance(P>0.05).2)Western blot results showed:the p-AMPK、RIP140、cleaved-caspase 3、Bax、p53 protein level of Metformin group、RIP140-overexpression group、RIP140-shRNA+Metformin group are increased,the Bcl-2 protein level is down,p-AMPK/AMPK、Bax/ Bcl-2 are increased on statistical significance(P<0.05),and total AMPK protein showd no statistical significance(P>0.05),compared with control group;compared with Metformin group,the protein level of p-AMPK、RIP140、cleaved-caspase 3、Bax、p53 in RIP140-shRNA+Metformin group decreased a little,and the Bcl-2 protein、p-AMPK/AMPK、Bax/ Bcl-2 also improved a little without statistical significance(P>0.05)Conclusion:1、Metformin dose-dependent increase in PGC-1α,the expression of RIP140,which inhibit the liver cancer cell proliferation play,apoptosis induction effect,and its mechanism may be related to activation of AMPK.2、RIP140 upregulation can promote apoptosis of liver cancer cells,downregulation can inhibit apoptosis of liver cancer cells,RIP140 may assume the role of tumor suppressor genes in the development of HCC.3、Metformin can increase RIP140 expression,which may activaed AMPK、caspase 3,regulated p53,Bax expression levels and the ratio of Bax / Bcl-2 and down-regulated Bcl-2 expression levels related,but this mechanism is not completely dependent of AMPK activation of apoptosis to regulate the expression of downstream target genes,there are other alternative pathway.